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FAQ

Tag-protein Purification Kit

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  1. I cannot purify epitope-tagged target proteins from my samples.
    If you can successfully confirm that the target protein was expressed rightly by other methods (e.g. western blotting), check whether the epitope-tagged target protein binds to anti-epitope tag antibody conjugated beads (tag beads) or not.
    → Briefly, after incubation the tag beads with the samples, please add SDS-PAGE sample buffer to tag beads, boil it for 5 minutes and perform SDS-PAGE or western blotting.
    If you cannot observe the target band as the results, please read the Q2.
    If you can obtain the target band as the results, please read the Q3.
  2. My epitope-tagged target protein does not bind to the tag beads.
    There are several possible causes as follows;
    -- Your target protein is insoluble or aggregated.
    -- Your lysis buffer contains guanidine and/or high concentrated urea. Some reagents can inhibit antigen-antibody reaction.
    → Please refer to “Additional information” in the data sheet for each PURIFICATION KIT. “Additional information” is about the propriety of usage of reagents contained in cell lysis buffer.
    -- Sometimes the compatibility between epitope-tags and target proteins affects to protein solubility.
    In such cases, try to change the location of epitope tag or use other epitope tags.
  3. My epitope-tagged target protein binds to tag beads, but I cannot elute the protein with peptide.
    Please try to prolong the incubation time or increase elution peptide volume.
    If your proteins are easy to be aggregated, please change buffer composition.
    e.g. Use lysate buffer for washing step instead of Wash Solution which is included in the PURIFICATION KIT.
    When you change composition of buffers, please refer to “Additional information” in the data sheet for each PURIFICATION KIT. “Additional information” is about the propriety of usage of reagents contained in cell lysis buffer.
  4. Which lysis buffer should I use?
    The buffer which contains usual detergent (e.g. NP-40 and Tween 20) and 0.1%-SDS can be used.
  5. Does the epitope tag location affect purification efficiency?
    In many cases, MBL’s PURIFICATION KITs can purify the epitope-tagged target proteins regardless of tag location.
    However, preliminary study is important in any cases.
  6. Can I elute the epitope-tagged target protein at 4℃ ?
    Most of MBL’s PURIFICATION KITs accept 4℃ in elution step. However, in His tagged Protein PURIFICATION KIT, the purification efficiency is reduced to almost halve if you elute the epitope-tagged target protein at 4℃ . If you have to perform elution step at 4℃ , please incubate His-tag beads with Elution Peptide Solution at 4℃ overnight before elution.
  7. Can I purify the epitope-tagged target protein from inclusion bodies of E. coli ?
    Some buffers which contain guanidine and/or high concentrated urea cannot be used for purification using MBL’s PURIFICATION KITs. Aggregated or insoluble proteins cannot be purifi ed, because such proteins cannot bind to tag beads.
    Therefore, please establish appropriate buffer condition with referring to “Additional information” in the data sheet for each PURIFICATION KIT. “Additional information” is about the propriety of usage of reagents contained in cell lysis buffer.
  8. Can I elute the epitope-tagged target protein using buffers other than Elution Peptide Solution?
    Yes you can. SDS-PAGE sample buffer, acid elution solution, and alkali elution solution can be used. However, the target proteins may lose protein activity and native conformation if you use such elution buffers.
    Acid elution solution: 0.1 M Glycine-HCl, pH 3.0 (Neutralize the elution immediately with 1 M Tris-HCl, pH 8.0)
    Alkali elution solution: 0.1M NH3, pH 11.3 (Neutralize the elution immediately with 1 N acetic acid)